RESUMO
BACKGROUND: Parasite traits associated with transmission success, such as the number of infective stages released from the host, are expected to be optimized by natural selection. However, in the trematode parasite Schistosoma mansoni, a key transmission trait, i.e. the number of cercariae larvae shed from infected Biomphalaria spp. snails, varies significantly within and between different parasite populations and selection experiments demonstrate that this variation has a strong genetic basis. In this study, we compared the transmission strategies of two laboratory schistosome population and their consequences for their snail host. METHODS: We infected inbred Biomphalaria glabrata snails using two S. mansoni parasite populations (SmBRE and SmLE), both isolated from Brazil and maintained in the laboratory for decades. We compared life history traits of these two parasite populations by quantifying sporocyst growth within infected snails (assayed using qPCR), output of cercaria larvae and impact on snail host physiological response (i.e. hemoglobin rate, laccase-like activity) and survival. RESULTS: We identified striking differences in virulence and transmission between the two studied parasite populations. SmBRE (low shedder (LS) parasite population) sheds very low numbers of cercariae and causes minimal impact on the snail physiological response (i.e. laccase-like activity, hemoglobin rate and snail survival). In contrast, SmLE (high shedder (HS) parasite population) sheds 8-fold more cercariae (mean ± SE cercariae per shedding: 284 ± 19 vs 2352 ± 113), causes high snail mortality and has strong impact on snail physiology. We found that HS sporocysts grow more rapidly inside the snail host, comprising up to 60% of cells within infected snails, compared to LS sporocysts, which comprised up to 31%. Cercarial production is strongly correlated to the number of S. mansoni sporocyst cells present within the snail host tissue, although the proportion of sporocyst cells alone does not explain the low cercarial shedding of SmBRE. CONCLUSIONS: We demonstrated the existence of alternative transmission strategies in the S. mansoni parasite consistent with trade-offs between parasite transmission and host survival: a "boom-bust" strategy characterized by high virulence, high transmission and short duration infections and a "slow and steady" strategy with low virulence, low transmission but long duration of snail host infections.
Assuntos
Biomphalaria/parasitologia , Schistosoma mansoni/fisiologia , Schistosoma mansoni/patogenicidade , Esquistossomose mansoni/parasitologia , Esquistossomose mansoni/transmissão , Animais , Biomphalaria/fisiologia , Brasil , Cercárias , Estudos de Coortes , Cricetinae , DNA de Helmintos/química , DNA de Helmintos/isolamento & purificação , Vetores de Doenças , Feminino , Hemoglobinas/análise , Hemolinfa/química , Hemolinfa/enzimologia , Humanos , Lacase/análise , Masculino , Mesocricetus , Reação em Cadeia da Polimerase Multiplex , Reação em Cadeia da Polimerase em Tempo Real , Schistosoma mansoni/crescimento & desenvolvimento , Razão de Masculinidade , VirulênciaRESUMO
Carbonic anhydrase (CA) is a ubiquitous metalloenzyme of great importance in several physiological processes. Due to its physiological importance and sensitivity to various pollutants, CA activity has been used as biomarker of aquatic contamination. Considering that in bivalves the sensitivity of CA to pollutants seems to be tissue-specific, we proposed here to analyze CA activity of hemolymph, gill and mantle of Crassostrea rhizophorae collected in two tropical Brazilian estuaries with different levels of anthropogenic impact, in dry and rainy season. We found increased carbonic anhydrase activity in hemolymph, gill and mantle of oysters collected in the Paraíba Estuary (a site of high anthropogenic impact) when compared to oysters from Mamanguape Estuary (inserted in an area of environmental preservation), especially in the rainy season. CA of hemolymph and gill were more sensitive than mantle CA to aquatic contamination. This study enhances the suitability of carbonic anhydrase activity for field biomarker applications with bivalves and brings new and relevant information on hemolymph carbonic anhydrase activity as biomarker of aquatic contamination.
Assuntos
Anidrases Carbônicas/metabolismo , Crassostrea/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Brânquias/enzimologia , Hemolinfa/enzimologia , Poluentes Químicos da Água/toxicidade , Animais , Brasil , Anidrases Carbônicas/genética , EstuáriosRESUMO
In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas' disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia.
Assuntos
Fosfatase Ácida/fisiologia , Catepsina D/fisiologia , Atresia Folicular , Hemípteros/enzimologia , Proteínas de Insetos/fisiologia , Insetos Vetores/enzimologia , Fosfatase Ácida/química , Animais , Catepsina D/química , Doença de Chagas/parasitologia , Corpo Adiposo/enzimologia , Feminino , Expressão Gênica , Hemípteros/parasitologia , Hemolinfa/enzimologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Insetos/química , Insetos Vetores/parasitologia , Células MCF-7 , Masculino , Especificidade de Órgãos , Ovário/enzimologia , Proteólise , Trypanosoma cruzi/fisiologia , Vitelinas/química , Vitelinas/metabolismoRESUMO
Several physiological processes can induce daily variations in aerobic metabolism. Lithodes santolla is a decapod crustacean of special concern since it is a sub-Antarctic species of commercial interest. The aim of this work was to study in L. santolla the daily variations in levels of enzymatic and non-enzymatic antioxidants, lipid peroxidation and protein oxidation, and haemolymphatic pH. Males of L. santolla of commercial size were randomly dissected every 4 h during a period of 24 h. Enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase and glutathione peroxidase were determined in samples of gills, muscle, hepatopancreas and haemolymph. Ascorbic acid, total glutathione, lipid peroxidation and protein oxidation were also determined in all tissues. Gills showed the highest enzymatic activity and hepatopancreas the highest concentration of non-enzymatic antioxidants. Maximum antioxidant activity was during the dark phase in gills and during the photophase in the haemolymph. Muscle showed significant daily variations, with peaks during the photophase and scotophase. Overall, an antioxidant protective mechanism is present in all tissues, where SOD and CAT represent the first line of defense. The defense mechanism in L. santolla seems to be more active during the dark phase, with slight differences among the analyzed tissues, indicating a higher metabolic rate.
Assuntos
Antioxidantes/metabolismo , Decápodes/fisiologia , Animais , Ácido Ascórbico/metabolismo , Catalase/metabolismo , Decápodes/enzimologia , Decápodes/metabolismo , Brânquias/enzimologia , Brânquias/metabolismo , Brânquias/patologia , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Hemolinfa/enzimologia , Hemolinfa/metabolismo , Hemolinfa/fisiologia , Hepatopâncreas/enzimologia , Hepatopâncreas/metabolismo , Hepatopâncreas/fisiologia , Concentração de Íons de Hidrogênio , Peroxidação de Lipídeos , Masculino , Músculos/enzimologia , Músculos/metabolismo , Músculos/fisiologia , Oxirredução , Superóxido Dismutase/metabolismoRESUMO
Introducción. Las úlceras crónicas son una afección con un impacto negativo importante en la calidad de vida de los pacientes y en el sistema de salud; la aparición de infecciones y su difícil manejo, así como la presencia de tejido necrótico, afectan el pronóstico de curación. La larvaterapia se presenta como una opción para el desbridamiento y el manejo de infecciones de úlceras crónicas. Objetivo. Evaluar la larvaterapia en heridas con poca carga de tejido necrótico y evaluar las excreciones, secreciones y la hemolinfa de las larvas, respecto a su contenido enzimático. Materiales y métodos. Se reporta una serie de tres casos clínicos con úlceras crónicas y poca carga de tejido necrótico, tratados con larvaterapia, y se evalúa su evolución por los índices PUSH (Pressure Ulcer Scale for Healing) y Wound Bed Score, así como el patrón electroforético y contenido enzimático por zimograma de las excreciones y secreciones, y de la hemolinfa de las larvas. Resultados. Con solo una aplicación de la larvaterapia se evidenció una mejoría del aspecto de la herida y en los puntajes evaluados; en el PUSH hubo una disminución de 2,3 puntos, en promedio, y con el Wound Bed Score, un incremento de 2,7, lo que demuestra una mejoría en ambas escalas. Conclusión. Se encontró una actividad enzimática diversa en su contenido de excreciones y secreciones, con predominio de actividad de la proteasa de tipo serina.
Introduction. Chronic leg ulcers are a burden for the health system and impact quality of life. The infections, the necrotic tissue and the difficult treatment affects the prognosis and healing time. Maggot therapy is presented as an acceptable alternative for the debridement and treatment of this pathology. Objective. The larval therapy was assessed on chronic leg ulcers with little necrotic tissue. Larval excretion and secretion (E/S) was characterized with respect to hemolymph (HL) enzymatic content. Materials and methods. Three patients with chronic leg ulcers and low necrotic tissue were treated with larval therapy and were assessed with the PUSH (pressure ulcer scale for healing) and Wound Bed Score. E/S and HL content was evaluated by SDS PAGE and zymogram. Results. The clinical aspect of the wounds showed improvement, and the scores demonstrated an average decrease of 2.3 for the PUSH and an average increase of 2.7 for the Wound Bed Score. A wide diversity of enzymatic activity in the E/S was demontrated with major activity belonging to serine protease family. Conclusions. Maggot therapy proved an effective treatment in cases with minimal tissue necrosis and can be considered a viable treatment option.
Assuntos
Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Terapia Biológica , Desbridamento/métodos , Pé Diabético/terapia , Proteínas de Insetos/análise , Larva/enzimologia , Peptídeo Hidrolases/análise , Úlcera Varicosa/terapia , Antibacterianos/uso terapêutico , Terapia Combinada , Pé Diabético/patologia , Dípteros/enzimologia , Dípteros/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Hemolinfa/enzimologia , Proteínas de Insetos , Necrose , Manejo da Dor , Peptídeo Hidrolases , Índice de Gravidade de Doença , Úlcera Varicosa/tratamento farmacológico , Úlcera Varicosa/patologia , CicatrizaçãoRESUMO
INTRODUCTION: Chronic leg ulcers are a burden for the health system and impact quality of life. The infections, the necrotic tissue and the difficult treatment affects the prognosis and healing time. Maggot therapy is presented as an acceptable alternative for the debridement and treatment of this pathology. OBJECTIVE: The larval therapy was assessed on chronic leg ulcers with little necrotic tissue. Larval excretion and secretion (E/S) was characterized with respect to hemolymph (HL) enzymatic content. Materials and methods. Three patients with chronic leg ulcers and low necrotic tissue were treated with larval therapy and were assessed with the PUSH (pressure ulcer scale for healing) and Wound Bed Score. E/S and HL content was evaluated by SDS PAGE and zymogram. RESULTS: The clinical aspect of the wounds showed improvement, and the scores demonstrated an average decrease of 2.3 for the PUSH and an average increase of 2.7 for the Wound Bed Score. A wide diversity of enzymatic activity in the E/S was demonstrated with major activity belonging to serine protease family. CONCLUSIONS: Maggot therapy proved an effective treatment in cases with minimal tissue necrosis and can be considered a viable treatment option.
Assuntos
Terapia Biológica , Desbridamento/métodos , Pé Diabético/terapia , Proteínas de Insetos/análise , Larva/enzimologia , Peptídeo Hidrolases/análise , Úlcera Varicosa/terapia , Adulto , Idoso , Animais , Antibacterianos/uso terapêutico , Terapia Combinada , Pé Diabético/patologia , Dípteros/enzimologia , Dípteros/crescimento & desenvolvimento , Eletroforese em Gel de Poliacrilamida , Feminino , Hemolinfa/enzimologia , Humanos , Proteínas de Insetos/metabolismo , Masculino , Necrose , Manejo da Dor , Peptídeo Hidrolases/metabolismo , Índice de Gravidade de Doença , Úlcera Varicosa/tratamento farmacológico , Úlcera Varicosa/patologia , CicatrizaçãoRESUMO
Lonomia achelous is a caterpillar distributed in southern Venezuela and in northern Brazil that causes an acute hemorrhagic syndrome in people who have contact with its bristles. The effect of the crude hemolymph and its chromatographic fractions (FDII, Lonomin V and Lonomin V-2) on extracellular matrix proteins was studied. The chromatographic fractions show activities similar to plasmin and urokinase. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both lonomins appear as a protein band of 25 kDa under reduced conditions. By exclusion chromatography, the molecular weights of Lonomin V and Lonomin V-2 were 26.5 and 24.5 kDa, respectively. Fibronectin, laminin and vitronectin were degraded by all venom components. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reduced conditions, shows that lonomins degrade fibronectin in four main fragments of 116, 60, 50 and 30 kDa. Molecular exclusion chromatography in native conditions shows that the molecular masses of these fragments are > or = 300, 62 and 27 kDa. The proteolytic effect of lonomins was abolished by benzamidine/HCl, iodoacetic acid and aprotinin. The extracellular matrix protein degradation together with the fibrino(geno)lytic activity of hemolymph and its fractions could explain, in part, the hemorrhagic syndrome, and the wound dehiscence in persons who have had contact with the L. achelous caterpillar.
Assuntos
Venenos de Artrópodes/farmacologia , Proteínas da Matriz Extracelular/efeitos dos fármacos , Hemolinfa/enzimologia , Mariposas/enzimologia , Serina Endopeptidases/farmacologia , Animais , Venenos de Artrópodes/isolamento & purificação , Cromatografia em Agarose , Fibronectinas/efeitos dos fármacos , Hemorragia/induzido quimicamente , Hemorragia/enzimologia , Hemostasia/efeitos dos fármacos , Hemostasia/fisiologia , Humanos , Laminina/efeitos dos fármacos , Serina Endopeptidases/isolamento & purificação , Vitronectina/efeitos dos fármacosRESUMO
Adenosine is an important signaling molecule for many cellular events. Adenosine deaminase (ADA) is a key enzyme for the control of extra- and intra-cellular levels of adenosine. Activity of ADA was detected in hemolymph of B. glabrata and its optimum assay conditions were determined experimentally. The pH variation from 6.2 to 7.8 caused no significant change in ADA activity. Using adenosine as a substrate, the apparent Km at pH 6.8 was 734 micromols.L(-1). Highest activity was found at 37 degrees C. Standard assay conditions were established as being 15 minutes of incubation time, 0.4 microL of pure hemolymph per assay, pH 6.8, and 37 degrees C. This enzyme showed activities of 834 +/- 67 micromol.min(-1).L(-1) (25 degrees C) and 2029 +/- 74 micromol.min(-1).L(-1) (37 degrees C), exceeding those in healthy human serum by 40 and 100 times, respectively. Higher incubation temperature caused a decrease in activity of 20% at 43 degres C or 70% at 50 degrees C for 15 minutes. The ADA lost from 26% to 78% of its activity when hemolymph was pre-incubated at 50 degrees C for 2 or 15 minutes, respectively. Since the ADA from hemolymph presented high levels, it can be concluded that in healthy and fed animals, adenosine is maintained at low concentrations. In addition, the small variation in activity over the 6.2 to 7.8 range of pH suggests that adenosine is maintained at low levels in hemolymph even under adverse conditions, in which the pH is altered.
Assuntos
Adenosina Desaminase/sangue , Biomphalaria/enzimologia , Hemolinfa/enzimologia , Adenosina Desaminase/metabolismo , Animais , Concentração de Íons de HidrogênioRESUMO
A adenosina é uma molécula sinalizadora de muitos eventos celulares. A adenosina desaminase (ADA) é enzima chave para o controle dos níveis intra e extra celulares de adenosina. A atividade da ADA foi detectada em hemolinfa de B. glabrata e suas condições ótimas de ensaio foram determinadas experimentalmente. A variação do pH de 6,2 até 7,8 não causou mudança significativa na atividade. O Km aparente foi de 734 µmoles L-1, usando adenosina como substrato. A maior atividade foi encontrada usando 37ºC como temperatura de incubação. As condições de ensaio padrão foram então estabelecidas como sendo 15 minutos de tempo de incubação, 0,4 µL de hemolinfa por ensaio, pH 6.8 e 37ºC de temperatura de incubação. A enzima apresentou atividades de 834 ± 67 µmols.min-1.L-1 (25ºC) e 2029 ± 74 µmols.min-1.L-1 (37ºC), em torno de 40 e 100 vezes maiores que os níveis encontrados em soro de humanos sadios. Em temperaturas superiores, essa atividade cai 20% a 43ºC e 70% a 50ºC, em 15 minutos. A ADA perde 26 a 78% de sua atividade quando a hemolinfa é pré-incubada a 50ºC de 2 a 15 minutos, respectivamente. Considerando os altos níveis de ADA encontrados pode-se inferir que, em animais sadios e alimentados, a adenosina é mantida em baixas concentrações na hemolinfa. Tendo a atividade da enzima permanecido constante frente à larga faixa de pH testada, sugere-se que a ADA pode atuar com eficiência mesmo em situações adversas que determinem variações no pH da hemolinfa.
Assuntos
Animais , Adenosina Desaminase/sangue , Biomphalaria/enzimologia , Hemolinfa/enzimologia , Adenosina Desaminase/metabolismo , Concentração de Íons de HidrogênioRESUMO
Phenoloxidase (monophenol, l-dopa: oxygen oxidoreductase, EC 1.14.18.1) is a multicopper oxidase, which plays an important role in melanin synthesis, necessary for defense against intruding microorganisms and parasites, wound healing and cuticle pigmentation. A phenoloxidase from the hemolymph of honey bee pupae exhibited an apparent molecular mass of 70 kDa, as estimated by gel filtration and SDS-PAGE. Optimal pH and temperature were 6.5 and 20 degrees C, respectively. Activity was fully stable for 30 min at 50 degrees C. Like phenoloxidases from the hemolymph of other insects, the honey bee enzyme was activated by trypsin and inhibited by protease inhibitors and phenylthiourea. Only high concentrations of sodium azide effectively inhibited the detected activity. A low concentration (5 microM) of Ca2+, Mg2+, and Mn2+ had a stimulatory effect on the activity. Single Michaelis-Menten curves were observed for l-dopa and dopamine oxidation, but the affinity of the enzyme for dopamine was greater than for L-dopa. Semiquantitative RT-PCR and Southern blot analysis using a 359 bp labeled probe, and quantification of the prophenoloxidase mRNA levels by real-time PCR showed increased amounts of transcripts in hemocytes and integument from young pupae injected with 20-hydroxyecdysone.
Assuntos
Abelhas/enzimologia , Catecol Oxidase/biossíntese , Ecdisterona/fisiologia , Precursores Enzimáticos/biossíntese , Monofenol Mono-Oxigenase/metabolismo , Animais , Abelhas/crescimento & desenvolvimento , Ecdisterona/farmacologia , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hemolinfa/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Metais/farmacologia , Pupa/enzimologia , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Azida Sódica/farmacologia , TemperaturaRESUMO
Occurrence and level of hydrolytic enzymatic activity (proteases, glycosidases, phosphatases, lipases, and esterases) were studied in oocytes, larvae, juveniles, and adult haemolymph of the Pacific oyster Crassostrea gigas. Samples were obtained as oocyte lysate supernatant, larval homogenate supernatant, juvenile homogenate supernatant, haemocyte lysate supernatant, and plasma. The presence of enzymes was demonstrated by colorimetric and lysoplate assay techniques. Between stages, significant differences in enzymatic activity determined by the colorimetric technique were found. Higher levels of enzymatic activity were found in the adult stage. Lysozyme-like activity was not found in oocytes, but was present in larvae, juveniles, and adults. In larvae, the highest lysozyme-like activity was in 3-d larvae. Juveniles had a 48-fold higher level of lysozyme-like activity, compared with 20-h larvae and was six-fold higher compared with 3-d larvae. In adults, lysozyme-like activity had a five-fold higher level in haemocyte lysate supernatant compared with plasma and was 98-fold higher compared with 20-h larvae. As determined with the API ZYM kit, 19 hydrolytic enzymatic activities were present, in oocytes, larvae, juveniles, and adult haemolymph of C. gigas. The presence of important lysozyme-like activity was confirmed from trochophora larvae (20 h) to adult stages.
Assuntos
Hemolinfa/enzimologia , Muramidase/sangue , Oócitos/enzimologia , Ostreidae/enzimologia , Fatores Etários , Animais , Proteínas Sanguíneas/isolamento & purificação , Colorimetria , Hidrólise , Larva/enzimologia , MéxicoRESUMO
Investigations on the effects of eicosanoid biosynthesis inhibitors on the hemocyte microaggregation and prophenoloxidase (proPO)-activating system in the hemolymph, parasitemia and mortality of Rhodnius prolixus infected with Trypanosoma rangeli were performed. Hemocoelic injection of live T. rangeli epimastigotes into fifth-instar larvae of R. prolixus that previously fed on blood containing an inhibitor of phospholipase A(2) (dexamethasone), a specific inhibitor of the cyclooxygenase pathway (indomethacin), and a non-selective lipoxygenase inhibitor (NDGA) (i) reduced the hemocyte microaggregation, (ii) attenuated the proPO system in the hemolymph and (iii) enhanced parasitemia and mortality induced by the parasite challenge in these insects. The effects obtained by dexamethasone administered orally were counteracted by inoculation of the insects with arachidonic acid. We suggest that the infectivity of T. rangeli can be increased by interference with the R. prolixus immune system. This is the first demonstration that the triatomine's immune responses to a parasite infection are modulated by a physiological system that includes eicosanoid biosynthesis.
Assuntos
Eicosanoides/antagonistas & inibidores , Eicosanoides/imunologia , Hemolinfa/imunologia , Insetos Vetores/imunologia , Rhodnius/imunologia , Serina Endopeptidases/efeitos dos fármacos , Tripanossomíase/imunologia , Animais , Ácido Araquidônico/metabolismo , Contagem de Células Sanguíneas , Dexametasona/farmacologia , Suscetibilidade a Doenças , Eicosanoides/biossíntese , Inibidores Enzimáticos/farmacologia , Hemócitos/imunologia , Hemolinfa/enzimologia , Indometacina/farmacologia , Insetos Vetores/enzimologia , Insetos Vetores/parasitologia , Masoprocol/farmacologia , Rhodnius/enzimologia , Rhodnius/parasitologia , Transdução de Sinais/imunologiaRESUMO
Investigations were carried out to compare aspects of the prophenoloxidase (proPO)-activating pathway in Rhodnius prolixus hemolymph in response to oral infection and inoculation of the insects with two developmental forms of Trypanosoma rangeli epimastigotes strain H14. In vivo experiments demonstrated that in control insects fed on uninfected blood, inoculation challenge with short epimastigotes resulted in high phenoloxidase (PO) activity. In contrast, previous feeding on blood containing either short or long epimastigotes was able to suppress the proPO activation induced by thoracic inoculation of the short forms. In vitro assays in the presence of short epimastigotes demonstrated that control hemolymph or hemolymph provided by insects previously fed on blood containing epimastigotes incubated with fat body homogenates from control insects significantly increased the PO activity. However, fat body homogenates from insects previously fed on blood containing epimastigotes, incubated with hemolymph taken from insects fed on control blood or blood infected with epimastigotes, drastically reduced the proPO activation. The proteolytic activity in the fat body homogenates of control insects was significantly higher than in those obtained from fat body extracts of insects previously fed on blood containing epimastigotes. These findings indicate that the reduction of the proteolytic activities in the fat body from insects fed on infected blood no longer allows a significant response of the proPO system against parasite challenge. It also provides a better understanding of T. rangeli infection in the vector and offer novel insights into basic immune processes in their invertebrate hosts.
Assuntos
Catecol Oxidase/metabolismo , Precursores Enzimáticos/metabolismo , Rhodnius/enzimologia , Rhodnius/parasitologia , Trypanosoma/fisiologia , Animais , Regulação para Baixo , Ativação Enzimática , Corpo Adiposo/enzimologia , Hemolinfa/enzimologia , Fatores de Tempo , Trypanosoma/crescimento & desenvolvimento , Tripsina/metabolismoRESUMO
The use of sequential measurements of hemolymph cholinesterase activities as a non-invasive biomarker of seasonal organophosphate/carbamate exposure was investigated for the tropical scallop, Euvola (Pecten) ziczac. Overall activities of both acetylcholinesterase and butyrylcholinesterase were relatively high compared to studies with bivalve tissues. Acute in vivo experiments showed inhibition of hemolymph acetylcholinesterase activity at concentrations of the organophosphate insecticide chlorpyrifos of 0.1, 1 and 10 ng l(-1). Monthly sampling of hemolymph from scallops at two sites in Bermuda over a 15 month period showed seasonal acetylcholinesterase and butyrylcholinesterase inhibition. Direct and indirect evidence suggests that this inhibition did not relate to biochemical or physiological changes associated with gonad maturation and spawning, but rather reflected diffuse contamination of the marine environment by cholinesterase inhibitors or increased bioavailability of such inhibitors at these times. Repetitive sampling of scallop hemolymph for cholinesterase activities represents a rapid, sensitive and non-invasive method for assessing seasonal, sublethal pesticide exposure in these commercially important bivalves and suggests a wider use in marine pollution monitoring.
Assuntos
Colinesterases/farmacologia , Monitoramento Ambiental/métodos , Moluscos , Praguicidas/efeitos adversos , Poluentes Químicos da Água/efeitos adversos , Animais , Bioensaio/métodos , Disponibilidade Biológica , Biomarcadores/análise , Hemolinfa/enzimologia , Praguicidas/análise , Poluentes Químicos da Água/análiseRESUMO
The clottable protein (CP) was isolated from white shrimp, Penaeus vannamei plasma as a 400-kDa protein that splits to two identical 200-kDa subunits when it is reduced with 2-ME. However, using DTT as reducing agent, four main bands were observed; two of them (179 and 125 kDa) had the same N-terminus sequence of the intact CP, indicating that most fragmentation occurs in the carboxy-terminus. The proteinase activity of reduced CP was detected using azoalbumin as substrate. Proteinase activity was only detected in the reduced, but not alkylated protein. Trypsin and papain, as well as soybean trypsin inhibitor and E64, were included for comparison. Proteolytic activity of reduced CP was inhibited by E64, but not by STI, indicating that such activity corresponds to a cysteine type proteinase.
Assuntos
Endopeptidases/metabolismo , Penaeidae/enzimologia , Animais , Endopeptidases/isolamento & purificação , Hemolinfa/enzimologia , Papaína/metabolismo , Tripsina/metabolismoRESUMO
Aminotransferases (GOT and GPT) activities in the hemolymph of Bradybaena similaris under experimental condition of starvation were studied. At the 10th day of starvation, GOT activity was 416.6% higher than that observed in the fed snails, being reduced and ranging values near to that shown by the control group onwards. GPT activity only varied significantly at the day-30 of starvation. The results were discussed.
Assuntos
Aspartato Aminotransferases/sangue , Hemolinfa/enzimologia , Caramujos/enzimologia , Inanição/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Análise de RegressãoRESUMO
Protease activities in the haemolymph and fat body in a bloodsucking insect, Rhodnius prolixus, infected with Trypanosoma rangeli, were investigated. After SDS-polyacrylamide gel electrophoresis containing gelatin as substrate, analysis of zymograms performed on samples of different tissues of controls and insects inoculated or orally infected with short or long epimastigotes of T. rangeli, demonstrated distinct patterns of protease activities: (i) proteases were detected in the haemolymph of insects which were fed on, or inoculated with, short epimastigotes of T. rangeli (39 kDa and 33 kDa, respectively), but they were not observed in the fat body taken from these insects; (ii) protease was also presented in the fat bodies derived from naive insects or controls inoculated with sterile phosphate-saline buffer (49 kDa), but it was not detected in the haemolymph of these insects; (iii) no protease activity was observed in both haemolymph and fat bodies taken from insects inoculated with, or fed on, long epimastigotes of T. rangeli. Furthermore, in short epimastigotes of T. rangeli extracts, three bands of the protease activities with apparent molecular weights of 297, 198 and 95 kDa were detected while long epimastigotes preparation presented only two bands of protease activities with molecular weights of 297 and 198 kDa. The proteases from the insect infected with T. rangeli and controls belong to the class of either metalloproteases or metal-activated enzymes since they are inhibited by 1,10-phenanthroline. The significance of these proteases in the insects infected with short epimastigotes of T. rangeli is discussed in relation to the success of the establishment of infection of these parasites in its vector, R. prolixus.
Assuntos
Metaloendopeptidases/metabolismo , Rhodnius/enzimologia , Rhodnius/parasitologia , Tripanossomíase/enzimologia , Tecido Adiposo/enzimologia , Animais , Hemolinfa/enzimologiaRESUMO
A polyclonal antibody (anti-HEX) was developed against a soluble N-acetylhexosaminidase (HEX) isolated from larval extracts of Boophilus microplus. Purified hexosaminidase was strongly inhibited by the IgG fraction of this antibody. The antibody inhibited the hexosaminidase activity of other sources, such as haemolymph and larval membranes. The antibody reacted with different antigens in the tick haemolymph, but did not recognize any antigen in saliva, as seen by immunoblot analysis. The anti-HEX was inoculated into fully engorged B. microplus females, resulting in a decrease in oviposition of approximately 26%, relative to the effect of pre-immune IgG. These data show the potential of the use of this tick enzyme as an antigen in vaccine development.
Assuntos
Anticorpos/imunologia , Doenças dos Bovinos/prevenção & controle , Infestações por Carrapato/veterinária , Carrapatos/fisiologia , beta-N-Acetil-Hexosaminidases/imunologia , Animais , Western Blotting/veterinária , Bovinos , Cromatografia em Agarose/veterinária , Cromatografia em Gel/veterinária , Compostos Cromogênicos/química , Eletroforese em Gel de Poliacrilamida/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Hemolinfa/enzimologia , Imunoglobulina G/imunologia , Oviposição/imunologia , Coelhos , Infestações por Carrapato/prevenção & controle , Carrapatos/imunologia , beta-N-Acetil-Hexosaminidases/análiseRESUMO
An aspartic proteinase precursor, herein named BYC (Boophilus Yolk pro-Cathepsin) was isolated from eggs of the hard tick, Boophilus microplus. As judged by electrophoresis on sodium dodecyl sulfate polyacrylamide slab gel (SDS-PAGE), purified BYC presented 2 bands of 54 and 49 kDa, bearing the same NH2-terminal amino acid sequence. By Western blot analysis, BYC was also found in the haemolymph, indicating an extraovarian site of synthesis. Several organs were incubated in culture medium with [35S]methionine, and only the gut and fat body showed synthesis of BYC polypeptides. Protein sequencing of both the NH2-terminal and an internal sequence obtained after cyanogen bromide (CNBr) cleavage of BYC revealed homology with several aspartic proteinase precursors. Incubation at pH 3.5 resulted in autoproteolysis of BYC, which produced the mature form of the enzyme, that displayed pepstatin-sensitive hydrolytic activity against haemoglobin. Western blot analysis using anti-BYC monoclonal antibodies showed proteolytic processing of BYC during embryogenesis and suggested activation of the enzyme during development. A role of BYC in degradation of vitellin, the major yolk protein of tick eggs, is discussed.
Assuntos
Ácido Aspártico Endopeptidases/isolamento & purificação , Precursores Enzimáticos/isolamento & purificação , Carrapatos/enzimologia , Tecido Adiposo/enzimologia , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/química , Western Blotting , Cromatografia DEAE-Celulose , Ovos , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/química , Feminino , Hemoglobinas/metabolismo , Hemolinfa/enzimologia , Intestinos/enzimologia , Túbulos de Malpighi/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Coelhos , Carrapatos/crescimento & desenvolvimentoRESUMO
Analysis of zymograms with SDS-polyacrilamide gel electrophoresis containing gelatin as substrate, and performed on samples of haemolymph or fat body taken from Rhodnius prolixus inoculated or not with Enterobacter cloacae, demonstrated distinct patterns of protease activities: (i) in the haemolymph two proteases were induced in insects inoculated with bacteria; (ii) two proteases were detected in the fat bodies derived from non-inoculated controls or insect inoculated with sterile culture medium; (iii) haemolymph and fat body had both the same apparent molecular weights proteases (46 and 56 kDa); and (iv) these enzymes were characterized as metallo-proteases. The association of these enzymes in Rhodnius infected with bacteria was discussed.